A great variety of microorganisms cause disease in humans. Bacterial infections are caused by gram-positive and gram-negative bacteria, spirochetes, mycobacteria, rickettsias, chlamydias and mycoplasmas. Yeast and systemic fungal pathogens are also a significant health problem in both the immunocompetent and the immunocompromised host. Cancer is another disease in humans.
Bacterial infections caused by gram-positive bacteria such as Streptococcus, Staphylococcus, Enterococcus, Bacillus, Corynebacterium, Listeria, Erysipelothrix, and Clostridium, and by gram-negative bacteria such as Haemophilus, Shigella, Vibrio cholerae, Neisseria and certain types of Escherichia coli, cause serious morbidity throughout the world. This, coupled with the emerging resistance shown by bacteria to currently used antibiotics, indicates the need for the development of bacterial vaccines to avoid these adverse consequences. Many current bacterial vaccine strategies involve the use of polysaccharide-comprising antigens associated with the bacterial cell wall (1, 3, 4, 11, 14, 15, 16, 23, 30, 31, 32, 34, 35, 37, 45, 46, 48).
A common feature for all bacteria, fungi and some protozoa is the presence of polysaccharides in and/or attached to their cell wall. Polysaccharides are an important architectural feature of the cell wall and contribute to the protection of microorganisms from attack by the immune system. Microbes have evolved a variety of different cell wall and cell wall-associated structures to avoid recognition and destruction by both the innate and adaptive arms of the host's immune system. For example, phagocytic cells of the innate immune system have specific cell surface receptors that recognize cell wall and cell wall-associated antigens. The CD14 receptor and Toll-like receptors (TLR) have been demonstrated to recognize peptidoglycan, lipoteichoic acid, and polysaccharide antigens; and in so doing, activate macrophages to phagocytose microorganisms (24, 38, 49). The adaptive arm of the immune system can also respond to these antigens, by generating antibodies that are specific to cell wall polysaccharide structures (29, 41).
Gram-positive and gram-negative bacteria are distinguished by their outer cell surface structure. The gram-positive cell wall is often devoid of lipids and is composed mostly of peptidoglycan, accessory polymers such as teichoic and/or teichuronic acid covalently linked to peptidoglycan, and protein. Peptidoglycan, also known as murein or mucopeptide, is composed of a polysaccharide backbone consisting of alternating repeats of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) with peptide side chains containing D- and L-amino acids. Gram-negative bacteria possess a cell wall composed of an outer membrane and a layer of peptidoglycan embedded between the outer membrane and an inner cytoplasmic membrane. The outer membrane contains lipopolysaccharide (LPS) as well as lipids and proteins. The LPS molecule is composed of a lipid A ‘head’ that is integrated into the outer membrane and a polysaccharide tail that extends outward from the outer membrane. The polysaccharide tail usually consists of a core oligopolysaccharide and an O-polysaccharide. The O-polysaccharide resembles peptidoglycan in having a basic repeating motif consisting of the alternating composition of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Also, capsular polysaccharides (CPS) and subcapsular polysaccharides are attached to the outer surface of gram-positive and gram-negative cell walls, and further serve to protect bacterial cells from recognition and engulfment by phagocytes.
Because the polysaccharide-containing components of the bacterial outer surface are specifically recognized by immune receptors, bacteria mutate their outer surface composition. For example, although cell wall polysaccharides are mainly composed of repeating (oligo)polysaccharide units, bacterial sub-strains vary this basic structure by having different modifications and/or ordering of the polysaccharide units. For instance, Group B Streptococci have a group antigen common to all serotypes (i.e., group polysaccharides (GPS)), but have type-specific capsular polysaccharides (CPS) that distinguish the major serotypes (types Ia, Ib, II, III, IV, V, VI, VII, and VIII). The structure of each of these various type-specific capsular polysaccharides has been characterized (19, 20, 21, 22, 43, 44, 47). Similarly, Streptococcus pneumoniae have a common group antigen (C-substance), but different type-specific capsular polysaccharides. Currently, there are 90 known serotypes of S. pneumoniae that are differentiated by their capsular polysaccharide coat (7, 49). Because of outer surface variation, bacterial types (or sub-strains) may be identified and classified according to type-specific antibodies. Thus, methods that can purify cell wall antigenic polysaccharides, either in their native state or as antigenic substructures, efficiently and simply, will greatly aid vaccine development.
Group and type-specific polysaccharides have been successfully used as targets for protective antibodies, where the production of these antibodies is provided by vaccines that stimulate active immunity. The antibodies generated in response to vaccination with vaccines having a type-specific polysaccharide component are also useful for providing passive immunity. Large-scale production of polysaccharides for use in vaccine manufacture requires adequate supplies of purified polysaccharides. Some methods (43, 45) for isolating capsular polysaccharides from certain bacterial cells rely on treatment of cells with the enzyme mutanolysin. Mutanolysin cleaves the bacterial cell wall which frees the cellular components. This procedure also involves treating cell lysate with additional enzymes to degrade proteins and nucleic acids. Other reported methods (50) for isolating polysaccharides from microorganisms rely on treatment of cells with hot phenol. Phenol separates the polysaccharides into the aqueous layer. The aqueous layer is concentrated by ultrafiltration to yield crude polysaccharide fractions which are then purified by chromatography. These methods are laborious and costly, as the necessity to eliminate toxic molecules associated with the cell wall (e.g., lipid A endotoxin, lipoteichoic acids and muramyl peptides) requires the use of expensive enzymes and complicated chromatography to degrade and remove toxins. More efficient, higher yielding and simpler means of obtaining purified polysaccharides are desirable.
Yet another effective method for isolating capsular polysaccharides (described in U.S. Pat. No. 6,248,570) relies upon base hydrolysis to extract CPS from cell wall components. This method allows for the degradation and removal of nucleic acids, proteins, and toxins from polysaccharides without the laborious and costly use of enzymes. Although this base hydrolysis method affords greater simplicity, efficiency, safety, and general applicability in relation to other previously reported methods, an even greater improvement of this method has been achieved in the present invention.
Advantages of the present invention include at least one of (1) simplicity, (2) an increase in molecule yield, (3) scalability (i.e., large scale production), (4) the purified polysaccharide can retain or be returned to its native antigenic form, and (5) DNA, RNA, and toxins are degraded in the hydrolysis steps and therefore are not present in appreciable amounts in the final product produced according to this invention.